Three dimensional electron microscopy (3DEM) is a set of techniques for determining the structures of large proteins and protein complexes. It has been shown that cryogenic EM (cryo-EM) has the ability to determine structures to high enough resolution to unambiguously place side chains in the EM density. Though cryo-EM has the potential for atomic resolution, a number of hurdles are present in order to achieve high-throughput structure determination of low-symmetry and no symmetry complexes. Here we propose to develop software tools for high-throughput high-resolution 3DEM. In the first aim, we propose to develop software tools for single particle 3DEM including software for automated specimen screening, automated data acquisition parameter optimization, and an infrastructure for processing helical samples. In the second aim, we propose to develop tools for tomographic data collection and processing including acquisition of dual axis tilt series, and developing and infrastructure for processing tomographic subvolumes. This research will benefit human health by facilitating the determination of protein structures that are relevant for human disease.